CASE REPORT Open Access

Case report: recurrent pituitary adenomahas increased load of somatic variantsRaitis Peculis1, Inga Balcere2,3, Ilze Radovica-Spalvina1, Ilze Konrade2,3, Olivija Caune2, Kaspars Megnis1,Vita Rovite1,4* , Janis Stukens5, Jurijs Nazarovs5, Austra Breiksa5, Aigars Kiecis2, Ivars Silamikelis1, Valdis Pirags4,5 andJanis Klovins1

Abstract

Background: Pituitary adenomas (PA) have an increased potential for relapse in one to 5 years after resection. Inthis study, we investigated the genetic differences in genomic DNA of primary and rapidly recurrent tumours in thesame patient to explain the causality mechanisms of PA recurrence.

Case presentation: The patient was a 69-year-old female with non-functional pituitary macroadenoma withextension into the left cavernous sinus (Knosp grade 2) who underwent craniotomy and partial resection in August2010. Two years later, the patient had prolonged tumour growth with an essential suprasellar extension (Knospgrade 2), and a second craniotomy with partial tumour resection was performed in September 2012. In bothtumours, the KI-67 level was below 1.5%. Exome sequencing via semiconductor sequencing of patient germlineDNA and somatic DNA from both tumours was performed. Tmap alignment and Platypus variant calling wereperformed followed by variant filtering and manual review with IGV software. We observed an increased load ofmissense variants in the recurrent PA tumour when compared to the original tumour. The number of detectedvariants increased from ten to 26 and potential clonal expansion of four variants was observed. Additionally,targeted SNP analysis revealed five rare missense SNPs with a potential impact on the function of the encodedproteins.

Conclusions: In this case study, an SNP located in HRAS is the most likely candidate inducing rapid PA progression.The relapsed PA tumour had a higher variation load and fast tumour recurrence in this patient could be caused byclonal expansion of the leftover tumour tissue.

Keywords: Recurrent pituitary adenoma, NFPA, Pituitary adenoma exome sequencing, Tumour variant analysis

BackgroundPituitary adenomas (PA) are neoplasms of the adenohy-pophyseal cells with benign characteristics but withoutclear markers for enhanced expansion or tumour re-growth after an operation. Clinically significant aden-omas are rare and affect about one in 1000 to 1300individuals [1, 2], although there are reports of theprevalence of up to one PA in 200 individuals [3]. Be-tween 35 and 40% of diagnosed PAs are non-functionaltumours (NFPA) that do not secrete hormones at de-tectable levels, while the rest (about 60–65%) are

hormone-secreting PAs [4]. Remission after surgery isreported to be 35.3–44.4% for NFPAs, 60.9% for somato-troph adenomas, 72.7% for corticotroph adenomas and61.7% for lactotroph adenomas. The highest recurrencerate has been reported at 1–5 years after surgery [5, 6].Both genetic and epigenetic factors are involved in the

development of PA [7]. However, the genetic causes ofthe majority of PA cases remain undiscovered, and thereason for this may be that a variety of somatic singlenucleotide variants (SNVs) can trigger the developmentof clinically significant PA. The Knudson two-hit hy-pothesis is one explanation for the tumourigenesis ofsporadic PA [8]. This is supported by the finding thatPA has a monoclonal origin [9].

© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: vita.rovite@biomed.lu.lv1Latvian Biomedical Research and Study Centre, Ratsupites str. 1-k1, RigaLV-1067, Latvia4University of Latvia, Raina blvd. 19, Riga LV-1586, LatviaFull list of author information is available at the end of the article

Peculis et al. BMC Endocrine Disorders (2020) 20:17 https://doi.org/10.1186/s12902-020-0493-x

Known causal genes for sporadic PA include GNAS,PI3KCA and USP8, as well as known familial PA genes(such as MEN1 and AIP), that often harbouring missenseand nonsense SNVs in sporadic tumours [10–15].Recently, several studies have been performed to

search for novel PA variants in genomic and tumourDNA pairs using whole-exome and genome sequen-cing. Newey et al. in 2013 sequenced the exomes ofseven NFPA and blood-derived DNA pairs. Theyfound low levels of somatic variants, which corres-pond to the benign nature of PA. The average novelvariant rate was 3.5 per adenoma (with a range of 1–7) and no overlapping mutated genes were found inthe follow-up group consisting of 24 PA cases [16]. Astudy performed in the East Asian population tookthe systematic approach of sequencing seven types ofPA including 20 NFPA samples. In that study, nooverlapping NFPA variants were found, but TRIP12and IARS had variants shared with adrenocorticotro-pic hormone (ACTH)-secreting PA and prolactin(PRL)-secreting PA, respectively, providing insightinto potential general genetic drivers of PA develop-ment. The median somatic novel variant rate perNFPA exome was 4.3 (range 0–13) [17]. A separatestudy identified a heterozygous missense variantc.4136G > T (p. Arg1379Leu) in cadherin-related 23(CDH23) in a family with inherited PA, and the genewas later screened in familial PA, sporadic PA andhealthy controls. The results revealed that four out of12 families (33%) and 15 out of 125 (12%) sporadicPA patients had variants in CDH23, providing yet an-other candidate for the development of PA [18]. Therole of potential drivers in the clonal evolution of PAcells is still to be elucidated.Many studies of malignant tumour types have

sought to gain insight into the process of carcinogen-esis using massively parallel genomics to assess sev-eral tumours from the same individual (primary,secondary, relapse tumour or metastasis), revealingmajor intratumoural heterogeneity that guides tumourevolution [19]. It has been shown that recurrent lungadenocarcinomas display a higher proportion of sub-clonal variants (40%) compared to primary tumourswithout relapse (17%) [20], indicating that the overallnovel variant load may increase recurrence potential.There are indications of clonal expansion in someforms of non-malignant neoplasms. For colorectal ad-enomas, it has been demonstrated that the same indi-vidual has heterogeneous methylation patterns inseveral independent tumours [21], and another studyhas shown a shift to malignant forms of usually non-metastatic dermatofibrosarcoma protuberans with apotential influence of therapy-induced variants andmore aggressive sub-clone prevalence [22].

In this study, we used a rapid recurrence case ofNFPA to gain insight into potential intratumouralgenetic evolution within the same individual using ex-ome sequencing of a tumour’s somatic DNA. Thepresented study is the first to report an exome scaleprofile of somatic variants in PA in a primary and re-current tumour and indicates the contributing factorsfor PA clonal evolution and tumour relapse.

Case presentationThe patient was a 69-year-old female who was diag-nosed with pituitary macro-adenoma in 2010. At thetime of diagnosis, the patient complained of a visualdisturbance. The investigation revealed bi-temporalhemianopsia, nervus opticus right side atrophy andleft side sub-atrophy.A hormonal investigation showed an NFPA. Pre-

operative magnetic resonance imaging (MRI) demon-strated an endo-, supra- and parasellar macroadenomawith extension into the left cavernous sinus (Knospgrade 2) with a total CC dimension of 3,86 cm and alarge suprasellar component (Fig. 1). In August 2010,the patient underwent surgery via craniotomy, and partialresection of the suprasellar part of the pituitary adenomawas performed. Before the surgery, the patient was en-rolled in national government-funded biobank - the Gen-ome Database of the Latvian population (LGDB) [23].During recruitment patient has signed two written in-formed consents (1) broad consent for LGDB for the useof the samples and data for health-related studies, and (2)project-specific consent for pituitary tumour studies. Bothwritten informed consents obtained from the patient andbiobank and pituitary tumour studies have been approvedby the Central Medical Ethics Committee of Latvia (proto-col No. 22.03.07/A7 and 01.29.1/28, respectively). Writteninformed consent for publication of the clinical detailsand/or clinical images was obtained from the patient. Acopy of the consent form is available for review by the Edi-tor of this journal.Immunohistochemical analysis of the tumour tissue

showed negative immunostaining for p53 protein expres-sion, PRL, growth hormone (GH), ACTH, thyroid-stimulating hormone (TSH), follicle-stimulating hormonebeta-subunit (FSH), somatostatin receptor 5 (SSTR5) andT-box transcription factor TBX19 (T-Pit); mild immuno-staining for POU domain class 1 transcription factor 1(PIT1); moderate immunostaining for steroidogenic factor1 (SF1); and strong positive immunostaining for luteinizinghormone beta subunit (LH), glycoprotein hormones alphasubunit (CGA), somatostatin receptor 2 (SSTR2) and arylhydrocarbon receptor-interacting protein (AIP). The distri-bution of cytokeratin-8 (CK8) was diffuse throughout thetumour, the KI-67 level was 1.5%. Haematoxylin and eosinstaining of the tumour is presented in Fig. 2. The

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patient developed secondary hypothyroidism and secondaryadrenal insufficiency after surgery what was substituted ac-cording to thyrotropic and corticotropic axis. She wasstarted on thyroxin and hydrocortisone supplementation.The first postoperative MRI was performed 3 months after

surgery. A clinical record indicates that MRI showed a largeresidual tumour at the left side of the cavernous sinus anda cystic structure that was also suprasellar. No images wereavailable in the electronic medical records of the patient forthis procedure. Subsequent radiotherapy was declined.

Fig. 1 The coronal (a) and sagittal (b) magnetic resonance images of pituitary macroadenoma with suprasellar extension into the left cavernoussinus (Knosp grade 2). The images were obtained before the first surgery in July 2010. The coronal (c) and sagittal (d) magnetic resonance imagesof pituitary macroadenoma with prolonged tumour growth with essential suprasellar and extension into both cavernous sinuses. The imageswere obtained before the second surgery in July 2012

Fig. 2 Haematoxylin and eosin staining of a primary and b recurrent formalin-fixed paraffin-embedded sections of the tumour, in 10×magnification. Both adenomas are composed of small-to-medium size chromophobic and in some places poorly basophilic, monomorphic,rounded cells with round nuclei, disperse nuclear chromatin and in some places with a well-developed nucleolus

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Two years later, an MRI of the pituitary gland (Fig. 1)revealed prolonged tumour growth with an essentialsuprasellar extension (Knosp grade 2). The patient wason thyroxin and hydrocortisone, and clinical evaluationrevealed NFPA with partial hypopituitarism after thefirst surgery. The patient underwent a second craniot-omy with partial tumour resection in September 2012.The immunohistochemistry analysis showed negativeimmunostaining for ACTH, TSH and T-Pit; mild immu-nostaining for PRL, GH, FSH, SSTR5 and PIT1; moder-ate immunostaining for LH, CGA and SF1; and strongpositive immunostaining for SSTR2 and AIP. The distri-bution of CK8 was diffuse throughout the tumour, andthe KI-67 level was 1%. After the second operation, thepatient continued medication as before. The visual fieldswere unchanged when compared with 2010.The patient was lost to follow-up, as she rarely attends

her family doctor and does not want further specialexamination. A detailed description of additional clinicaldata, materials, methods and timeline following theCARE guidelines are presented in Additional file 1.

Exome sequencing resultsFinal average exome (defined by UCSC Genome Browser(GRCh37/hg19) [24] interval file in Additional file 2)coverage was 60.5 ± 42.7X for white blood cells (WBC)derived DNA, 70.9 ± 210.7X for the exome of the firsttumour and 84.8 ± 273.9X for the exome of the secondtumour (for additional coverage, alignment and qualitymetrics see Additional file 3).The results of the exome-wide search for missense and

nonsense variants are shown in Table 1. In total, 26 mis-sense and zero non-sense variants were found in theexomes from the PA tumours of this patient. Ten vari-ants were present in the first and second tumour butnot in the germline DNA derived from patients WBC.Two of those variants showed evidence for statisticallysignificant clonal expansion after the first surgery(C11orf57 from 5.3% (4 reads at 76X depth) to 24.3% (17reads at 70X) and MED24 from 3.2% (2 reads at 62X) to22.2% (14 reads at 63X). An additional 16 variants werefound in the second tumour but not in the exome of theWBC or in the exome of the first tumour. However, itshould be mentioned that six (KIAA1841, DMXL1, AK9,PTPRK, PLEKHA7 and ITGAX) of these 16 positionshad lower than 10X coverage in the first tumour sequen-cing. Additionally, two genetic variants were representedin the germline DNA derived from WBC by one sequen-cing read of 53 (1,9%) and 70 (1,4%), respectively, andhave an expanded fraction of mutant alleles in theexomes from the PA samples.Gene ontology analysis revealed that three of the genes

are associated with the RNA binding process and two ofthem, ALKBH8 and AHNAK, contain a variant in the

first tumour. RGS14 and C5 are involved in the G pro-tein alpha signalling pathway, and APC and PCDHGA1are involved in the Wnt signalling pathway, but theproducts of ENPP1 and PTPRK possess phosphatase ac-tivity. Three variants have been previously reported inCOSMIC database PCDHGA1 (COSV65295567) in threecases - one prostate, one endometrium and one large in-testine tumours. CLMN (COSV54187254) has been re-ported in one cervical cancer sample and ZNF320(COSV67089305) in one thyroid carcinoma sample.We also performed a targeted analysis of SNPs located

in a gene set compiled from the literature on PA genet-ics, tumour suppression and genome and exome sequen-cing. By searching the literature with the keywords“pituitary adenoma genetics”, “pituitary adenoma exomesequencing”, “pituitary adenoma gwas” and “tumoursuppressor genes” on 15th of May 2018, a list of 403genes was compiled (Additional file 4). Targeted SNPanalysis was performed in this region which encom-passes 47,370,914 base pairs (including introns, whichare sparsely represented in exome data) (Fig. 3). Twohundred fifty-two missense SNPs were found in 133genes. SIFT and Polyphen prediction algorithms rated23 missense SNPs both “deleterious” and “probably dam-aging” or “possibly damaging”. CDH23 was the only genethat has two SNPs (rs45583140, rs1227049) recognizedby both algorithms as severely affecting protein function.A frequency filter with minor allele frequency thresh-

old 1% (gnomAD database, non-Finnish European popu-lation) left five SNPs as the top candidates possiblycontributing towards characteristics and development ofPA (Table 2). Four SNPs had “start-loss”, “stop-gain”and “stop-loss” consequences, but all have high popula-tion frequencies. CNVs detection using CoNIFER v0.2.2[25] yielded negative results.

Discussion and conclusionsHere we present the first study performing exome sequen-cing of recurrent PA from the same patient. Although theremission rate of NFPA after surgery is 44,4% [5], to thebest of our knowledge, exome or genome analysis of a re-current pituitary tumour pair is not yet published. At thesame time, there are studies on clonal expansion andintra-tumoural heterogeneity performed for a multitude ofother tumour types [19, 20, 22]. Our results indicate thatrapid recurrence of PA is led by an increase of novel vari-ant load in a benign tumour that might be explained byclonal selection of leftover tumour tissue. We also wouldlike to notice that the patient did not receive radiotherapyduring any stage of the treatment that would stimulateadditional mutagenesis.The number of variants in primary PA is low, is con-

sistent with the literature reported in other exome se-quencing studies on PA [16, 26] and corresponds to the

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mainly non-metastatic phenotype of PA. In spite of therelatively benign nature of PA, the regrowth in 2 yearsafter the first surgery indicates the proliferative potentialof the tumour cells in the studied patient. Previous epi-demiological studies have shown that recurrence of PAis most likely to happen during the 5 year period aftersurgery [5]. Our study indicates that a large proportionof SNVs from the first tumour was also present in thesecond tumour. This might indicate that, at least in thispatient, regrowth occurred from leftover cells that weremissed when resecting the primary tumour. Whetherthis finding can be generalized for all recurrent PAsshould be explored with more extensive studies. We alsoobserved an increased missense variant burden in thesecond PA when compared to the first tumour. This

increase in variant content can be explained by a dimin-ished ability to control DNA replication or influence ofsome of the SNVs that can serve as “drivers” for clonalexpansion when novel “passenger” variants are acquired.This has been observed frequently in other malignantcancers [27, 28], and there is some indication in similarprocesses in more neutral tumours [22], but this aspecthas not been widely studied in benign tumours. Alterna-tively, if not all of the PA tumour mass was removedduring the first surgery (in this particular case, leftovertissue was detected during an MRI scan 3 months aftersurgery), it implies that either the original tumour wasnot monoclonal, or the accumulation of variants mighthave led to the development of a group of heterogeneousclones. However, the monoclonality of PA has been

Table 1 Missense variants from exome sequencing of WBC and two consecutive PA derived DNA samples

Location Ref /Altallele

Gene CDSposition

Proteinposition

Aminoacids

SIFT PolyPhen Locus coverage/alternative variant depth

WBC 1st tumour 2nd tumour

Somatic variantsof the bothtumours

5:140712358a G/A PCDHGA1 2107 703 V/I 0.18 0.021 41/0 40/34 (85%) 30/12 (40%)

11:107375677 A/T ALKBH8 1702 568 C/S 0.43 0 41/0 80/27 (34%) 43/13 (30%)

11:111951148 T/A C11orf57 99 33 D/E 0 0.721 22/0 76/4 (5.3%) 70/17 (24%)

11:62301128 C/T AHNAK 761 254 G/E 0.18 0.999 94/0 64/29 (45%) 38/13 (34%)

14:95669606a A/G CLMN 2080 694 C/R 0.03 0 36/0 151/51 (34%) 260/75 (29%)

16:2855123 G/T PRSS41 – – – – – 105/0 73/11 (15%) 58/14 (24%)

17:38189695 T/G MED24 631 211 I/L 0.07 0.003 26/0 62/2 (3.2%) 63/14 (22%)

19:53384666a C/A ZNF320 713 238 S/I 1 0.005 55/0 9/1 (11%) 27/10 (37%)

20:23731308 G/T CST1 196 66 R/S 0 0.985 53/0 252/103 (41%) 241/125 (52%)

X:63444842 C/G ASB12 689 230 C/S 0.02 0.977 112/0 651/228 (35%) 632/234 (37%)

Somatic variantsof the secondtumour

2:11593766 T/C E2F6 97 33 N/D 0 0.132 125/0 150/0 117/13 (11%)

2:61349274 T/C KIAA1841 2134 712 F/L 0.13 0.001 25/0 7/0 12/6 (50%)

3:100058013 G/C NIT2 90 30 E/D 0.06 0.005 22/0 34/0 38/6 (16%)

5:112178502 T/C APC 7211 2404 M/T 0.54 0 86/0 18/0 30/7 (23%)

5:118552606 A/C DMXL1 7937 2646 D/A 0 0.234 22/0 4/0 6/5 (83%)

5:176797955 A/C RGS14 1177 393 T/P 0 0.962 1/0 15/0 42/10 (24%)

6:109480584 A/C CEP57L1 935 312 D/A 0.88 0.08 30/0 11/0 20/6 (30%)

6:109983834 G/T AK9 364 122 Q/K 0.06 0.738 37/0 1/0 69/19 (28%)

6:128326254 C/T PTPRK 2499 833 M/I 0.02 0.024 90/0 3/0 16/7 (44%)

8:17417949 A/G SLC7A2 1411 471 R/G 0.41 0 133/0 13/0 33/8 (24%)

9:123751971 G/A C5 3029 1010 A/V 0.21 0.713 75/0 25/0 23/8 (35%)

10:120801963 A/C EIF3A 3069 1023 D/E 0.12 0.005 75/0 21/0 43/15 (35%)

11:16812380 C/T PLEKHA7 3017 1006 G/D 0.12 0.575 57/0 8/0 32/6 (19%)

11:77911754 G/A USP35 1097 366 G/E 0.02 0.212 74/0 13/0 20/7 (35%)

12:113730866 G/A TPCN1 2457 819 M/I 0.17 0.053 88/0 70/0 25/10 (40%)

16:31371749 G/C ITGAX 826 276 A/P 0 0.999 15/0 7/0 12/7 (58%)

Variants withexpansion

4:85707175 C/T WDFY3 4019 1340 R/Q 0 0.787 53/1 (1.9%) 37/14 (38%) 23/10 (43%)

6:132171199 A/G ENPP1 383 128 E/G 0.38 0.003 70/1 (1.4%) 75/2 (2.7%) 34/10 (29%)aCosmic database variant (5:140712358, PCDHGA1, 703 V/I, COSV65295567; 14:95669606, CLMN, 694C/R, COSV54187254; 19:53384666, ZNF320,238S/I, COSV67089305)

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widely accepted [9, 16, 29]. Shared variants betweenthe tumour samples in our study also provide a highlikelihood of PA recurrence rather than the develop-ment of a separate novel neoplasm. Additionally, wedid not observe any cell lineage change or significantalteration of hormone production in presented pa-tient’s case. There are reports that in rare cases shiftfrom clinically silent PA to hormonally active tu-mours is observed [30–32], however, these do not in-clude exact tracing according to transcription factorsdefined by the WHO classification of 2017. To fur-ther assess potential shifts in cell lineage of operatedrecurrent PA cases comprehensive research of signifi-cant number of relapse PAs including T-pit, SF1,PIT-1 immuno-staining is needed.It is unlikely that the variants identified are the cause

of the tumours either alone or in a compound fashion,and the most likely explanation is that these are somatic“passenger” variants. Four genes (WDFY3, ENPP1,C11orf57 and MED24) with a low amount of the mu-tated reads in the germline DNA or the first tumour

could be attributed to the allele-specific replication ofthe germline and/or the first tumour during librarypreparation.The increased number of variants in the second tumour

could indicate a decreased ability of the tumour cells torepair DNA damage as proliferation increases and variantsaccumulate. It could also show that the tumour is trans-forming or being transformed by surgery towards a rela-tively hypermutated state with multiple disruptionscontributing to the aggressiveness of the tumour. Al-though this “hypermutated” PA state is lower than thenovel variant rate in hypermutated malignant tumours[33], it is significantly more than the average novel variantrate per PA exome of other studies [16, 17, 34].A targeted study of genes compiled from the litera-

ture on PA genetics and tumourigenesis revealed sev-eral interesting facts. CDH23 contained two SNPs(rs45583140, MAF in non-Finnish Europeans = 3.6%,rs1227049, MAF in non-Finnish Europeans = 17.2%)that are missense and predicted to be damaging tothe protein function. CDH23 encodes cadherin 23,which is involved in cell to cell adhesion [35] andhearing mechanism [36]. Cadherin 23 is localized atcell contact points, suggesting involvement in contactinhibition, and it is highly expressed in breast cancercompared to normal tissue [35]. Additionally, theCDH23 variant p.Arg1379Leu was found in a PA-affected family, and further screening showed that33% of familial PA patients and 125 of sporadic PApatients carry functional variants compared to 0.8% ofcontrols [18]. Therefore, it is possible that the twoidentified compound heterozygous CDH23 missenseSNPs (a population chance of 0.6%) could contributeto the quick PA regrowth in the patient studied viadisrupted contact inhibition.The five other SNPs identified in the targeted gene

search are located in ZFYVE26, JPH2, OR5M1, NPTXRand HRAS. All SNPs were in the heterozygous state.Three of the genes JPH2, OR5M1 and NPTXR have hada somatic variant (although different position) in previ-ous PA exome sequencing studies in the literature [26,37], and further functional studies are needed to evaluatethe role of these gene variants on pituitary cell function-ality. Recurrence of deleterious rare SNVs across differ-ent PA sequencing studies could indicate that they couldbe part of Knudson’s two hit hypothesis in PA, especiallywhen taking into account other genetic and/ or environ-mental factors. ZFYVE26 has been shown to harboursomatic mutations in hereditary non- polyposis colorec-tal cancer and is expressed in hepatocellular adenocar-cinoma) [38], but the potential relation to PAdevelopment is discussable. HRAS is widely describedproto-oncogene which predominates in head and necksquamous cell carcinoma [39] but also is found in the

Fig. 3 Targeted SNP search and filtering steps

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salivary duct [40], bladder urothelial carcinoma andacute myeloid leukaemia [39]. Having been implicated inother carcinogenesis cases, HRAS could be a novel can-didate that influences PA. A cosmic somatic variant(COSV54248437) found in our PA patient has been pre-viously found in cervix cancer [41]. Together with bothprediction algorithms assigning a negative impact onprotein functionality and the virtual absence in the gen-eral population, this could be one of the prime candi-dates that causes development or rapid growth of thetumour in our study.This study has some limitations. One limitation is that

exome sequencing may miss important variants outsidethe coding regions that may contribute to PA develop-ment. Whole-genome sequencing would overcome thislimitation. Similarly, although we had good sequencingcoverage in our samples, increased depth would providemore precision for variants identified in a low percent-age of reads. Additional sequencing, however, is not pos-sible due to the low amount of initial tumour materialavailable and the division of this material for the clinicalarchive and other research activities. We also did notperform Sanger sequencing validation as in other cancerstudies where the overall variant percentage in thetumour somatic material could be low, as these may notbe detectable by Sanger sequencing [17, 26, 37].In conclusion, we show that in this relapse case, the

regrowth of PA is accompanied by the increase of thetumour novel variant load, which could be caused byclonal expansion of the tumour leftover tissue.

Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s12902-020-0493-x.

Additional file 1. Timeline following the CARE guidelines. Additionalclinical data. A detailed description of Materials and Methods used in thestudy.

Additional file 2. Interval file of HG19 exome coordinates. Thisreference exome version was used to search for variants and CNVs.

Additional file 3. Document file (30 pages) describing alignment qualitymetrics for each sample (10 pages per sample).

Additional file 4. Excel spreadsheet with the list of genes used fortargeted PA genetic marker search.

AbbreviationsACTH: Adrenocorticotropic hormone; DNA: Deoxyribonucleic acid;IGV: Integrative genomics viewer; LGDB: Genome Database of Latvianpopulation; MAF: Minor allele frequency; MRI: Magnetic resonance imaging;NFPA: Non-functional pituitary adenoma; PA: Pituitary adenoma;RNA: Ribonucleic acid; SNP: Single nucleotide polymorphism; SNV: Singlenucleotide variant; WBC: White blood cell

AcknowledgementsThe authors acknowledge the Latvian Biomedical Research and Study Centreand the Genome Database of the Latvian Population for providinginfrastructure, biological material and data.

Authors’ contributionsAll authors have read and approved the final version of the manuscript. RPparticipated in the study design, performed data analysis and manuscriptwriting. IB performed patient enrolment in the study, clinical dataobtainment and description. IRS performed library preparation andsequencing. IK participated in the study design and manuscript editing. OCperformed clinical data obtainment and description. KM isolated DNA fromtissue samples. VR performed manuscript writing and submission. JN and ABperformed histopathological staining. AK analysed the MRI scans. JSperformed surgery and somatic tumour material obtainment. IS participatedin data analysis. VP participated in the study design and manuscript editing.JK was the project leader, performed study design and manuscript editing.

FundingThe research of the primary and recurrent pituitary tumour case wassupported by the European Regional Development Fund Measure 1.1.1.1“Industry-Driven Research” within the project “Molecular markers of pituitarytumour development, progression and therapy response” (Project no. 1.1.1.1/16/A/066).

Availability of data and materialsThe datasets used and/or analysed during the current study are availablefrom the corresponding author on reasonable request.

Ethics approval and consent to participateThe patient was recruited to the LGDB, a government-funded national bio-bank, by an endocrinologist at Pauls Stradins Clinical University Hospital, Riga,Latvia in 2010. LGDB is a central institution for human genetic material bio-banking for research needs in Latvia [23]. Two written informed consentswere obtained from the patient, broad consent for LGDB for use of biologicalmaterial and medical data for human health and hereditary research, andproject-specific consent with approval of the use of biological material andclinical data in research related to pituitary tumours that included all proce-dures and analyses described in this manuscript. In addition, upon requestfrom the journal, a specific written informed consent to publish the resultsas a case report was obtained prior to publication. Both the biobank studydesign and PA research study and both written informed consents obtained

Table 2 Candidate variants from targeted SNP analysis

Locus Gene Minor allele Aa change SNP code SIFT PolyPhen gnomAD NFE AF

chr 14:68274264 ZFYVE26 T 246 A/D rs367929006 del (0) possibly dmg (0.79) 0

chr 20:42788571 JPH2 C 286 T/A rs144022614 del (0) possibly dmg (0.45) 0.14%

chr11:56380836 OR5M1 T 48 L/Q rs183262731 del (0) probably dmg (1) 0.55%

chr22:39222627 NPTXR A 326 R/W rs34637063 del (0) possibly dmg (0.83) 0.59%

chr11:533515 HRAS A 130 A/S COSV54248437 del (0.04) possibly dmg (0.71) –

chr10:73571765a CDH23 C 3130 F/L rs45583140/ COSV56481145 del (0.01) probably dmg (0.99) 3.6%

10:73434888a CDH23 C 495 G/A rs1227049 del (0) probably dmg (1) 17.2%

Aa amino acid, SNP single nucleotide polymorphism, NFE non-Finnish European, AF allele frequency, del deleterious, dmg damagingacompound population frequency 0.6%

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from the patient have been approved by the Central Medical Ethics Commit-tee of Latvia (protocol No. 22.03.07/A7 and 01.29.1/28, respectively). The sam-ple collection and research process comply with the Declaration of Helsinki(in particular parts A and B) and the Declaration of Taipei.

Consent for publicationThe patient has consented to the national biobank Genome Database of theLatvian population design approved by the Central Medical EthicsCommittee of Latvia (protocol No. 22.03.07/A7) and pituitary adenomaresearch approved by the Central Medical Ethics Committee of Latvia(protocol No. 01.29.1/28). Both consents imply the use of the samples forhealth research, which includes reporting and confidentiality topics. TheInstitutional Review Board of Latvian Biomedical Research and Study Centre(Scientific Council) has evaluated the compliance of obtained consents andresults of the study and has approved this study to be published as casereport. Specifically, informed consent was obtained also from the patient forthe specific publication of this manuscript.

Competing interestsThe authors declare that they have no competing interests.

Author details1Latvian Biomedical Research and Study Centre, Ratsupites str. 1-k1, RigaLV-1067, Latvia. 2Riga East Clinical University Hospital, Hipokrata str. 2, RigaLV-1038, Latvia. 3Riga Stradins University, Dzirciema str. 16, Riga LV-1007,Latvia. 4University of Latvia, Raina blvd. 19, Riga LV-1586, Latvia. 5PaulsStradins Clinical University Hospital, Pilsonu str. 13, Riga LV-1002, Latvia.

Received: 13 March 2019 Accepted: 16 January 2020

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